The present invention is concerned with a process for producing Alpha-Interferon (IFN-.alpha.) in which chromatography on immunosorbents, namely on anti-interferon antibodies, is avoided in favour of anion exchange chromatography, the protein itself and the use thereof. The new process avoids (a) any virus contamination of the final product which might occur, and (b) a bottleneck in the production process caused by the use of antibody column.
Interferons are proteins naturally occurring in the body which have antiviral, antiproliferative and immunoregulatory activity. The antiviral effect is achieved not by a direct influence on the viruses themselves, but by an activity on their target cells in the sense of a protection against the virus infection. The interferons can exert objectifiable effects on cancer tumours, which make them suitable for use in cancer therapy, and they can influence the immune system of the body on that, for example, they activate macrophages and NK cells and intensify the expression of various immunologically significant constituents of the cell membrane.
Human interferons (alpha, beta and gamma) can today be prepared in a microbiological manner thanks to recombinant DNA technology in amounts which cannot be made available by isolation from natural material (leucocytes, fibroblasts, lymphocytes) and purification in spite of the greatest efforts.
This technology has opened a way for the intensive clinical testing and wide therapeutic use of interferons by providing an adequate supply of the active substances.
Details of the cloning of interferon-cDNA and the direct expression thereof, especially in E. coli, have in the meantime been the subject of many publications. Thus, for example, the preparation of recombinant interferons is known, for example, from Nature, 295 (1982), 503-508, Nature, 284 (1980), 316-320, Nature, 290 (1981), 20-26, Nucleic Acids Res., 8 (1980), 4057-4074, as well as from European Patents Nos. 32134, 43980 and 211 148.
Since the recombinant interferons are of microbial origin (e.g., they are preferably derived from E. coli), after their isolation from the microorganism or from the culture medium they are initially still contaminated by a series of microbial impurities, the presence of which is prohibitive for a therapeutic use of the thus-produced interferons. The purification of the recombinant material therefore plays a particularly important role. A multitude of different methods, especially chromatography, have hitherto been used and combined with one another for the purification of recombinant interferons. Above all, chromatography on immunoadsorbents, namely on anti-interferon antibodies, has proved to be a valuable aid. Thus, the purification of recombinant human leucocyte interferon (HuIFN-.alpha.) by means of monoclonal antibodies has been described, for example, by Staehelin et al. (J.Biol.Chem., 256 (1981), 9750-9754) and by Secher et al. (Nature, 285 (1980), 446-450). Having regard to the high specificity of these immunoadsorbents it must be assumed from this that the thus-purified material is practically free from contaminating substances and has a high degree of purity.
In the case of the purification of larger amounts of recombinant IFN-.alpha. by means of monoclonal antibodies it has, however, been found that the purified material contains not only interferon fragments (interferon in which a part of the terminal amino acid sequence is missing), but also interferon oligomers, for example, dimers. These undesirable by-products prompted the inclusion of additional chromatography steps in the purification process.
A method for producing IFN-.alpha. using immunoaffinity chromatography followed by copper chelate chromatography and cation exchange chromatography has been described in Chimia, 40 (1986) 408-412. However, the use of immunoaffinity chromatography may be accompanied by the problem of mouse immunoglobulin and virus contamination in the final product. Mouse immunoglobulin contamination could occur due to trace amounts of immobilized antibody being eluted with the final product. Viral contamination could occur due to bovine serum being used in the production of the monoclonal antibodies. Additionally, it has been found that this method is limited in scale since several runs on the antibody column are needed for one full batch.